Maximal activities of key enzymes of glutaminolysis, glycolysis, Krebs cycle and pentose-phosphate pathway of several tissues in mature and aged rats

1. The maximum activities of some key enzymes, which provide a quantitative indices of flux through several important pathways have been measured in brain, liver, muscle, white and brown adipose tissue and lymphocytes of mature and aged rats. 2. The results were expressed as mumol/min per g fresh weight and nmol/min per mg protein. 3. On the both basis, as compared to mature rats, hexokinase activity is decreased in brown adipose tissue and increased in soleus muscle. 4. Glucose-6-phosphate dehydrogenase activity is decreased in most tissues and increased in brain. 5. Citrate synthase activity, which provides a qualitative index of the Krebs cycle, is decreased in white adipose tissues and lymphocytes. 6. Glutaminase activity is decreased in brain, white and brown adipose tissues but is increased in lymphocytes.     

Maximum activities of key enzymes of glycolysis, glutaminolysis, pentose phosphate pathway and tricarboxylic acid cycle in normal, neoplastic and suppressed cells.

1. Maximal activities of some key enzymes of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle and glutaminolysis were measured in homogenates from a variety of normal, neoplastic and suppressed cells. 2. The relative activities of hexokinase and 6-phosphofructokinase suggest that, particularly in neoplastic cells, in which the capacity for glucose transport is high, hexokinase could approach saturation in respect to intracellular glucose; consequently, hexokinase and phosphofructokinase could play an important role in the regulation of glycolytic flux in these cells. 3. The activity of pyruvate kinase is considerably higher in tumorigenic cells than in non-tumorigenic cells and higher in metastatic cells than in tumorigenic cells: for non-tumorigenic cells the activities range from 28.4 to 574, for tumorigenic cells from 899 to 1280, and for metastatic cells from 1590 to 1627 nmol/min per mg of protein. 4. The ratio of pyruvate kinase activity to 2 x phosphofructokinase activity is very high in neoplastic cells. The mean is 22.4 for neoplastic cells, whereas for muscle from 60 different animals it is only 3.8. 5. Both citrate synthase and isocitrate dehydrogenase activities are present in non-neoplastic and neoplastic cells, suggesting that the full complement of tricarboxylic-acid-cycle enzymes are present in these latter cells. 6. In neoplastic cells, the activity of glutaminase is similar to or greater than that of hexokinase, which suggests that glutamine may be as important as glucose for energy generation in these cells.     

The content of pentose-cycle intermediates in liver in starved, fed ad libitum and meal-fed rats.

Liver content of pentose-cycle intermediates and the activity of the three major cytoplasmic NADPH-producing enzymes and pentose-cycle enzymes were measured in three dietary states: 48 h-starved rats, rats fed on a standard diet ad libitum, and rats meal-fed with a low-fat high-carbohydrate diet. Measured tissue contents of pentose-cycle intermediates in starved liver were: 6-phosphogluconate, 4.7 +/- 0.5 nmol/g; ribulose 5-P, 3.7 +/- 0.5 nmol/g; xylulose 5-P, 4.3 +/- 0.4 nmol/g; sedoheptulose 7-P, 25.5 +/- 1.3 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 30.6 +/- 0.7 nmol/g. These values were in good agreement with values calculated from fructose 6-P and free glyceraldehyde 3-P, assuming the major transketolase, transaldolase, ribulose-5-P 3-epimerase and ribose-5-P isomerase reactions were all in near-equilibrium. Similar results were found in animals fed ad libitum. These relationships were not valid in animals fed on a low-fat high-carbohydrate diet, with tissue contents of metabolites in some cases being more than an order of magnitude higher than the calculated values. Measured tissue contents of pentose-cycle intermediates in these animals were: 6-phosphogluconate, 124.2 +/- 13.9 nmol/g; ribulose 5-P, 44.8 +/- 7.1 nmol/g; xylulose 5-P, 77.2 +/- 9.4 nmol/g; sedoheptulose 7-P, 129.9 +/- 10.1 nmol/g; and combined sedoheptulose 7-P and ribose 5-P, 157.0 +/- 11.3 nmol/g. In all animals, regardless of dietary state, tissue content of erythrose 4-P was less than 2 nmol/ml. Liver activities of glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase were increased from 3.5 +/- 0.9 mumol/g and 7.3 +/- 0.5 mumol/min per g in starved animals to 13.2 +/- 1.1 and 10.5 +/- 0.7 mumol/min per g in low-fat high-carbohydrate-fed animals. Despite these changes, the activities of transaldolase (3.4 +/- 0.3 mumol/min per g), transketolase (7.8 +/- 0.2 mumol/min per g) and ribulose-5-P 3-epimerase (7.5 +/- 0.4 mumol/min per g) were not increased in meal-fed animals above those observed in starved animals (3.4 +/- 0.2, 7.1 +/- 0.3 and 8.6 +/- 0.4 mumol/min per g respectively). The increase in the activity of oxidative pentose-cycle enzymes in the absence of any change in the non-oxidative pentose cycle appeared to contribute to the observed disequilibrium in the pentose cycle in animals meal fed on a low-fat high-carbohydrate diet.     

A comparison of the properties of fructose 1,6-diphosphatase, and the activities of other key enzymes of carbohydrate metabolism, in the livers of embryonic and adult rat, sheep and domestic fowl

1. The activities of some key enzymes of glycolysis and gluconeogenesis were measured in embryonic chick, sheep and rat livers. 2. In chicken the activities of hexokinase, phosphofructokinase and pyruvate kinase are low, but those of glucose 6-phosphatase and fructose diphosphatase are very high; the converse situation exists in the rat (Burch et al. 1963), but in sheep the activities of both phosphofructokinase and fructose diphosphatase are high, and the activities of hexokinase and glucose 6-phosphatase are low. These findings are discussed in relation to carbohydrate metabolism in these embryonic livers. 3. The regulatory properties of fructose diphosphatase from the embryonic livers of these three species were compared with the properties of the enzymes from adult animals. The inhibitions by AMP and fructose diphosphate and the effects of Mg(2+) and pH on the activities of adult and foetal fructose diphosphatase are almost identical. 4. It is concluded that regulatory properties are characteristic of fructose diphosphatase from embryonic and adult tissue, and the importance of this in relation to enzyme development is discussed.     

METABOLIC CONTROL MECHANISMS IN MAMMALIAN SYSTEMS

Abstract— The regulation by thyroid hormone of the activities of hexokinase (ATP: D-hexose 6-phosphotransferase; EC 2.7.1.1), phosphofructokinase (ATP: D-fructose-6- phosphate 1-phosphotransferase; EC 2.7.1.11) and pyruvate kinase (ATP: pyruvate phosphotransferase; EC 2.7.1.40) has been investigated in the soluble fractions of the cerebral cortex and cerebellum of the rat. Ontogenetic studies on these key glycolytic enzymes demonstrated marked increases in the normal cerebral cortex between 1 day and 1 yr of age; less pronounced increases in enzyme activities were noted in the normal cerebellum. Neonatal thyroidectomy, induced by treatment of 1-day-old rats with 100 μCi of ¹³¹I, ied to an impairment of body and brain growth and inhibited the developmental increases in hexokinase, phosphofructokinase and pyruvate kinase in both the cerebral cortex and cerebellum. Whereas 50 μCi of ¹³¹I had little or no effect on these brain enzymes, 200 μCi of the radioisotope markedly inhibited (35–65 per cent) the developmental increases of the various enzyme activities investigated. When administration of the radioisotope was delayed for 20 days after birth, little or no inhibition of the development of brain glycolytic enzymes was observed. Whereas treatment of normal neonatal animals with L-tri-iodothyronine had no significant effect on the activities of cerebro-cortical and cerebellar glycolytic enzymes, the hormone increased their activities in young cretinous rats. However, when the initiation of tri-iodothyronine treatment was delayed until neonatally thyroidectomized rats had reached adulthood, this hormone failed to produce any appreciable change in enzyme activity. Our results indicate that thyroid hormone exerts an important regulatory influence on the activities of hexokinase, phosphofructokinase and pyruvate kinase in the developing cerebral cortex and cerebellum.     

Effect of citrate on the activities of 6-phosphofructokinase from nervous and muscle tissues from different animals and its relationships to the regulation of glycolysis.

1. Citrate inhibits the activities of phosphofructokinase from muscles and nervous tissues from different animals across the Animal Kingdom except for the insects. The enzymes from the flight muscle of nine different insects and the cerebral ganglion of the locust were investigated: no inhibition by citrate was observed. Inhibition was observed with the enzymes from both aerobic (e.g. pectoral muscle of pigeon) and anaerobic (e.g. fish muscle, pectoral muscle of the game birds) muscles. It is suggested that this inhibition is of physiological importance in decreasing the rate of glucose utilization in skeletal muscle of animals during starvation and/or prolonged exercise. 2. The rates of glucose utilization by the sartorius and gastrocnemius muscles of the frog were markedly decreased by ketone bodies. The latter elevated the glucose 6-phosphate and citrate contents of the gastrocnemius muscle, indicating that citrate inhibition of phosphofructokinase could be, in part, responsible for the decreased rate of glycolysis. 3. These findings provide evidence that the concept of the glucose-fatty acid-ketone-body cycle involves both aerobic and anaerobic skeletal muscle and nervous tissue from a wide range of animals except the insects. In the latter the concept of the cycle may not be applicable.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Glycolytic enzymes in mammalian spermatozoa. Activities and stabilities of hexokinase and phosphofructokinase in various fractions from sperm homogenates

1. Methods of homogenizing suspensions of washed mammalian spermatozoa were studied. The most useful methods were those using sonication and those using a French press. 2. Hexokinase, phosphofructokinase, glucose phosphate isomerase and adenosine triphosphatase activities in ram, bull and boar spermatozoa were investigated by using these two homogenization methods. Glucose phosphate isomerase, representative of soluble cytoplasmic material, was very readily extracted and remained entirely in the supernatant after centrifugation at 145000g for 60min. In contrast, the other three activities were less easily extracted and were sedimented in various proportions under the described conditions of centrifugation. 3. Attempts to obtain subcellular fractions from sperm homogenates by ;classical' methods failed, owing apparently to the inhomogeneity of subcellular particles in the homogenates. It is concluded that, after removal of sperm heads, the only meaningful fractionation is a separation of spermatozoal material which sediments at 145000g during 60min from that which does not. 4. The stabilities of hexokinase and phosphofructokinase activities in bull, boar and ram sperm homogenates were investigated. Hexokinases showed very little dependence on the various environments tested, whereas the optimum conditions for phosphofructokinase stability were: a minimum of sonication, the presence of phosphate ions and of a thiol-group protectant, and a pH7.5. Activities of hexokinase, phosphofructokinase and glucose phosphate isomerase per sperm cell were compared with published data on rates of fructolysis by spermatozoa; the potential catalytic activities were shown to be considerably in excess of these rates. However, phosphofructokinase may be the rate-limiting enzyme of glycolysis in vivo in bull and ram spermatozoa.     

Fuel utilization in colonocytes of the rat.

In incubated colonocytes isolated from rat colons, the rates of utilization O2, glucose or glutamine were linear with respect to time for over 30 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 30 min. Glutamine, n-butyrate or ketone bodies were the only substrates that caused increases in O2 consumption by isolated incubated colonocytes. The maximum activity of hexokinase in colonic mucosa is similar to that of 6-phosphofructokinase. Starvation of the donor animal decreased the activities of hexokinase and 6-phosphofructokinase, whereas it increased those of glucose-6-phosphatase and fructose-bisphosphatase. Isolated incubated colonocytes utilized glucose at about 6.8 mumol/min per g dry wt., with lactate accounting for 83% of glucose removed. These rates were not affected by the addition of glutamine, acetoacetate or n-butyrate, and starvation of the donor animal. Isolated incubated colonocytes utilized glutamine at about 5.5 mumol/min per g dry wt., which is about 21% of the maximum activity of glutaminase. The major end-products of glutamine metabolism were glutamate, aspartate, alanine and ammonia. Starvation of the donor animal decreased the rate of glutamine utilization by colonocytes, which is accompanied by a decrease in glutamate formation and in the maximum activity of glutaminase. Isolated incubated colonocytes utilized acetoacetate at about 3.5 mumol/min per g dry wt. This rate was not markedly affected by addition of glucose or by starvation of the donor animal. When colonocytes were incubated with n-butyrate, both acetoacetate and 3-hydroxybutyrate were formed, with the latter accounting for only about 19% of total ketones produced.     

Correlation of enzymatic,metabolic, and behavioral deficits in thiamin deficiency and its reversal

To clarify the enzymatic mechanisms of brain damage inthiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced¹⁴CO₂ and¹⁴C-acetylcholine from [U-¹⁴C]glucose at rates similar to controls under resting conditions, their K⁺-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60–65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3.) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30–32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.Thus, changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis. A more sustained deficit in the pentose pathway enzyme, transketolase, may relate to the anatomical abnormalities that accompany thiamin deficiency.Dedicated to Henry McIlwain.     

The effects of calcium ions on the activities of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from various muscles

1. The effects of Ca²⁺ on the activities and regulatory properties of trehalase, hexokinase, phosphofructokinase, fructose diphosphatase and pyruvate kinase from vertebrate red and white muscle and insect fibrillar and non-fibrillar muscle have been investigated. These muscles were selected because of the possible difference in the role of glycolysis in energy production in the vertebrate muscles, and the possible difference in the role of Ca²⁺ in the control of contraction in the two types of insect muscle. An increase in Ca²⁺ concentration from 0.001μm to 10μm did not modify the activities nor did it modify the regulatory properties of these enzymes from these various muscles. 2. Concentrations of Ca²⁺ above 0.1mm inhibited the activities of hexokinase and phosphofructokinase from the different muscles. It has been suggested that this inhibition may provide the basis for a theory of regulation of glycolysis (Margreth et al., 1967). If phosphofructokinase is located within the sarcoplasmic reticulum, its activity will be inhibited when the muscle is at rest, but the release of Ca²⁺ from the reticulum during contraction will lead to a stimulation of its activity and hence an increase in glycolytic flux. The distribution of hexokinase and phosphofructokinase in the various cell fractions of these muscles was very variable. In particular, both enzymes were present almost exclusively in the 100000g supernatant fraction in the extracts of insect flight muscles. Thus there is no correlation between the properties of the enzymes and their distribution in muscle. 3. It is concluded that Ca²⁺ does not control the activities of the important regulatory enzymes of glycolysis in muscle. It is suggested that in some muscles the sensitivity of the control mechanism at the level of phosphofructokinase to changes in the concentration of AMP may be increased by a process known as `substrate-cycling'.     

Activity and androgenic control of glycolytic enzymes in the epididymis and epididymal spermatozoa of the rat.

1. Procedures were developed for the extraction and assay of glycolytic enzymes from the epididymis and epididymal spermatozoa of the rat. 2. The epididymis was separated into four segments for analysis. When rendered free of spermatozoa by efferent duct ligation, regional differences in enzyme activity were apparent. Phosphofructokinase, glycerol phosphate dehydrogenase and glucose 6-phosphate dehydrogenase were more active in the proximal regions of the epididymis, whereas hexokinase, lactate dehydrogenase and phosphorylase were more active in the distal segment. These enzymes were less active in the epididymis of castrated animals and less difference was apparent between the proximal and distal segments. However, the corpus epididymidis from castrated rats had lower activities of almost all enzymes compared with other epididymal segments. 3. Spermatozoa required sonication to obtain satisfactory enzyme release. Glycolytic enzymes were more active in spermatozoa than in epididymal tissue, being more than 10 times as active in the case of hexokinase, phosphoglycerate kinase and phosphoglycerate mutase. 4. The specific activities of a number of enzymes in the epididymis were dependent on the androgen status of the animal. These included hexokinase, phosphofructokinase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, glycerol phosphate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and phosphorylase. 5. The caput and cauda epididymidis differed in the extent to which enzyme activities changed in response to an altered androgen status. The most notable examples were hexokinase, phosphofructokinase, aldolase, phosphoglycerate kinase, 6-phosphogluconate dehydrogenase and phosphorylase.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

3-Mercaptopyruvate sulfurtransferase activity in guinea pig and rat tissues

Activities of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) in guinea pig tissues were determined and compared with those in the corresponding rat tissues. The activities in guinea pig tissues were found to be very low. The activity in the liver was 145.9 mumol pyruvate formed per g of fresh tissue per 15 min. This was 1/50 of the activity in the rat liver. Activities in the kidney and brain were 1/100 of the corresponding rat tissues. Those in the erythrocyte and heart were negligibly low and far less than 1/1000 of these tissues in the rat. Similarities between the guinea pig and patients with beta-mercaptolactate-cysteine disulfiduria are discussed.     

Activities of glucokinase and hexokinase in mammalian and avian livers.

A radiochemical assay for glucokinase activity was developed for use in high-speed supernatants of liver. The maximum activities of glucokinase ranged from 0.4 to 3.8 mumol/min per g fresh wt. at 30 degrees C in some avian and mammalian livers, including pigeon, guinea pig and man, in which previous reports indicated zero activities. The reported maximum rates of hepatic glycogen synthesis in livers of rat and man in vivo are similar to the calculated glucokinase activities at 10mM-glucose; therefore glucokinase activity should not limit glycogen synthesis from glucose.     

Carbohydrate metabolism in human skeletal muscle during exercise is not regulated by G-1,6-P2

Glucose 1,6-bisphosphate (G-1,6-P2) is a potent activator of phosphofructokinase (PFK) and an inhibitor of hexokinase in vitro. It has been suggested that increases in G-1,6-P2 are a main means by which PFK can achieve significant catalytic function in vivo despite falling pH and that increases in G-1,6-P2 will inhibit hexokinase in vivo. The purpose of the present study was to determine whether contraction-induced changes in flux through PFK and hexokinase are associated with changes in G-1,6-P2 in skeletal muscle. Ten men performed bicycle exercise for 10 min at 40 and 75% of maximal O2 uptake (VO2max) and to fatigue [4.8 +/- 0.6 (SE) min] at 100% VO2max. Biopsies were obtained from the quadriceps femoris muscle at rest and after each work load and analyzed for G-1,6-P2. G-1,6-P2 averaged 111 +/- 13 mumol/kg dry wt at rest and 121 +/- 16, 123 +/- 15, and 123 +/- 11 mumol/kg dry wt after the low-, moderate-, and high-intensity exercise bouts, respectively (P less than 0.05 for all means vs. rest). Flux through PFK was estimated to increase exponentially as the exercise intensity increased and muscle pH decreased at the higher work loads, whereas flux through hexokinase was estimated to increase during exercise at 40 and 75% VO2max but decrease sharply at 100% VO2max. These data demonstrate that flux through neither PFK nor hexokinase is mediated by changes in G-1,6-P2 in human skeletal muscle during short-term dynamic exercise.     

Maximum activities and properties of glucose 6-phosphatase in muscles from vertebrates and invertebrates.

1. The maximum catalytic activities of glucose 6-phosphatase were measured in a large number of muscles from vertebrates and invertebrates. The activities range from less than 0.1 to 8.0 mumol/min per g fresh wt. at 30 degrees C: the highest activity, observed in the flight muscle of the wasp (Vespa vulgaris), is similar to that in rat liver. The hydrolytic activity was shown to be specific towards glucose 6-phosphate. 2. The pH optimum was 6.8 and the Km was approx. 0.6 mM (flight muscle of a moth). 3. Almost all of the glucose 6-phosphatase activity from extracts of the flight muscle of a moth and the pectoral muscle of a pigeon were recovered in the cytosolic fraction (i.e. 150,000 g supernatant). 4. During development of the locust (Schistocerca gregaria), the activity of the phosphatase in the flight muscle increased during the first 3 days after the final moult. 5. The activity of glucose 6-phosphatase from insect and avian muscle was separated from that of non-specific phosphatase on a Bio-Gel P-100 column. 6. For the activities from 63 muscles, there was a strong positive correlation between those of glucose 6-phosphatase and hexokinase, but no correlation between the activities of glucose 6-phosphatase and fructose bisphosphatase. It is suggested that the role of glucose 6-phosphate in muscle is either to produce glucose from glucose 6-phosphate derived from glycogen or to provide the enzymic basis for a substrate ("futile") cycle between glucose and glucose 6-phosphatase in muscle to improve the sensitivity of the mechanism that regulates the rate of glucose phosphorylation.     

Pathway of starch breakdown in photosynthetic tissues of Pisum sativum.

1. The aim of this work was to discover the pathway of starch breakdown in the photosynthetic tissues of Pisum sativum. 2. Measurements of the starch in the leaves of plants grown in photoperiods of 12 or 18 h showed that starch, synthesized in the light, was rapidly metabolized in the dark at rates of 0.04--0.06 mumol glucose/min per g fresh weight. 3. The maximum catalytic activities of alpha-amylase, beta-amylase, hexokinase, alpha-glucan phosphorylase and phosphoglucomutase in extracts of leaves showed no diurnal variation in either photoperiod, and exceeded estimates of the rate of net starch breakdown in the dark. 4. Studies with intact chloroplasts, isolated from young shoots and from leaves, indicated that pea chloroplasts do not contain significant activities of alpha-amylase, beta-amylase and hexokinase, although some of the latter may be attached to the outside of the chloroplast envelope. These studies also showed that pea chloroplasts contained sufficient alpha-glucan phosphorylase and phosphoglucomutase to mediate the observed rates of starch breakdown. 5. It is proposed that starch breakdown in pea chloroplasts is phosphorolytic.     

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.     

Regulation of pathways of glucose metabolism in kidney. The effect of experimental diabetes on the activity of the pentose phosphate pathway and the glucuronate-xylulose pathway.

Measurements have been made of the activities of enzymes of the pentose phosphate pathway, the glucuronate-xylulose pathway, hexokinase and phosphofructokinase in kidney of diabetic and normal rats. The activities of these enzymes keep pace with kidney growth, remaining constant per gram tissue but showing a marked increase on the basis of total activity per 100 g body wt. The formation of ¹⁴CO₂ from [1-¹⁴C]glucose and [6-¹⁴C]glucose by kidney slices from diabetic rats was decreased to approximately half the control value; evidence was obtained for an equivalent dilution of the glucose 6-phosphate pool. Correction of the ¹⁴CO₂ yields for the change in specific activity of glucose 6-phosphate yielded values consistent with the enzyme profile. Calculations from specific yields of ¹⁴CO₂ provided evidence for an increased flux of glucose via the pentose phosphate pathway in the kidney in diabetes. The results are discussed in relation to kidney hypertrophy in diabetes and the requirement for ribose 5-phosphate and NADPH for biosynthetic reactions and in relation to the thickening of the basement membrane in diabetes. These results are in accord with the concept of glucose overutilization by non-insulin-requiring tissues.